Temperature-sensitive specific polypeptide and method for non-destructive separation of exosomes based on temperature difference specificity

Abstract

The present invention relates to the technical field of separation of biological substances, and in particular to a temperature-sensitive specific polypeptide and a method for non-destructive separation of exosomes based on temperature difference specificity. In the present invention, a temperature-sensitive specific polypeptide having an amino acid sequence as shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 is provided; nanomagnetic microspheres are modified with the temperature-sensitive specific polypeptide and then mixed with a liquid containing exosomes, so that the exosomes are specifically bound to the temperature-sensitive specific polypeptide, thereby being able to separate the exosomes by magnetic force.

Description

Technical Field

[0001] The present invention relates to the technical field of biological material separation, and in particular to a temperature-sensitive specific polypeptide and a method for non-destructive separation of exosomes based on temperature difference specificity. Background Art

[0002] Exosomes are bilayer phospholipid membrane vesicles containing complex RNA and proteins. They play an important role in both physiological and pathological processes. Researchers have now found that they have great application potential in treatment and detection. High-purity exosomes are the basis for exosome research and application. How to effectively separate exosomes has always been the focus of scientific researchers. At present, the main exosome separation technologies include ultracentrifugation, ultrafiltration, polymer precipitation, size exclusion chromatography based on physical separation methods, and affinity separation methods based on biochemical separation methods.

[0003] Separation methods based on physical properties such as density and size, such as ultracentrifugation, are commonly used methods for exosome isolation. This method is based on the density gradient differences of various substances in body fluids, and uses centrifugal force to achieve the purpose of separating and enriching exosomes. Ultracentrifugation is relatively simple, not contaminated by separation reagents, and a large number of exosomes are obtained. It is still widely used. However, the centrifugation instrument required for this method is expensive, the sample volume is large, it takes a long time, and the purity of the isolated exosomes is not high.

[0004] The ultrafiltration method utilizes the specific size of exosomes and uses ultrafine nanofiltration membranes with different cut-off pore sizes to retain substances of specific sizes on the ultrafiltration membrane, and small particle size substances are filtered to the other side of the membrane. The ultrafiltration method is simple to operate and can concentrate exosome samples. It is low-cost and fast. The patent application with application number CN202310735921.1 discloses a technical solution for separating and purifying exosomes, in which the use of ultrasonic nanofiltration method may cause mechanical damage to the exosomes during pressurization or vibration, destroying their active structure; and the filter membrane is easily blocked by macromolecular substances, causing the filter membrane to break due to excessive pressure.

[0005] A large number of non-exosome particles with the same physical properties such as size and density will cause the exosomes obtained by physical separation methods to contain a large amount of contamination, such as vesicle-like apoptotic bodies, extracellular vesicles, as well as exosomes, polymers, lipoproteins, etc.

[0006] Affinity separation is a separation method that uses the specific interaction force between molecular pairs to covalently modify the molecules on the solid phase carrier to capture the other paired molecule. Using this principle to separate exosomes is to covalently modify the binding molecules of specific markers on the surface of exosomes, such as proteins, peptides or aptamers, on the surface of solid phase carriers (such as magnetic beads or microspheres). After incubation and binding with exosomes, the exosomes are separated and eluted from the liquid phase to achieve the purpose of separation and enrichment. The advantages of this method are high specificity, simple operation, and higher purity of exosomes separated compared to other separation methods. However, after exosomes bind to ligand molecules, extreme or complex dissociation conditions are required. For example, after immune antibodies bind to exosome marker proteins (CD63, CD9, CD81, etc.), they need to be carried out at a pH of about 3.0. Exosomes with complete structures cannot be obtained, their functionality will be lost, and there is a risk of protein-like substances damaging activity or aggregation. In the technical solution of the patent application with application number CN201580066077.1, based on the specific binding of TIM protein to phosphatidylserine, a high concentration of EDTA-2Na needs to be added to chelate the calcium ions that act as bridges between molecules so that the exosomes can be eluted. However, free ligands may affect subsequent use, and further separation will result in low separation efficiency.

[0007] In summary, the current lack of an efficient and specific non-destructive isolation method for exosomes has seriously hindered the development of exosome-related research and applications. Summary of the invention

[0008] Based on this, the present invention provides a temperature-sensitive specific polypeptide and a method for non-destructive separation of exosomes based on temperature difference specificity, which at least solves one problem in the prior art.

[0009] In a first aspect, the present invention provides a temperature-sensitive specific polypeptide, whose amino acid sequence is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3.

[0010] Thermosensitive specific polypeptide is a polypeptide that can specifically bind to the exosome surface marker. The secondary structure of the polypeptide is regulated by temperature, so that it can specifically bind to the exosome surface marker protein at low temperature (2~8℃); at high temperature (25~37℃), it will dissociate from the exosome surface marker protein, and its affinity is temperature sensitive. The amino acid sequence of the thermosensitive specific polypeptide is shown in Table 1, wherein the polypeptide corresponding to SEQ ID No.1 is a CD63 protein-specific polypeptide; the polypeptide corresponding to SEQ ID No.2 is a CD9 protein-specific polypeptide; and the polypeptide corresponding to SEQ ID No.3 is a CD81 protein-specific polypeptide. The polypeptide corresponding to SEQ ID No.1 specifically binds to CD63 protein (one of the exosome surface specific marker proteins) under low temperature conditions (2~8℃) and dissociates at higher temperatures (25~37℃). The polypeptide corresponding to SEQ ID No.2 specifically binds to CD9 protein (one of the exosome surface specific marker proteins) under low temperature conditions (2~8℃) and dissociates at higher temperatures (25~37℃). The polypeptide corresponding to SEQ ID No.3 specifically binds to CD81 protein (one of the specific marker proteins on the surface of exosomes) under low temperature conditions (2~8°C) and dissociates at higher temperatures (25~37°C).

[0011] Table 1 Amino acid sequences of temperature-sensitive specific peptides

[0012]

[0013] In a second aspect, the present invention provides the use of the temperature-sensitive specific polypeptide in isolating exosomes.

[0014] In a third aspect, the present invention provides a method for non-destructive separation of exosomes based on temperature difference specificity, which comprises the following steps:

[0015] Modifying nanomagnetic microspheres with temperature-sensitive specific polypeptides to obtain polypeptide-modified nanomagnetic microspheres;

[0016] The liquid containing exosomes is mixed with the polypeptide-modified nanomagnetic microspheres, and incubated at 2-8°C to allow the exosomes to bind to the polypeptide-modified nanomagnetic microspheres. Under the action of the magnetic field, the polypeptide-modified nanomagnetic microspheres capture and separate the exosomes from the liquid, and non-specific adsorbed substances are removed by washing. Thereafter, the liquid is incubated at 25-37°C to allow the exosomes to dissociate from the polypeptide-modified nanomagnetic microspheres, and the polypeptide-modified nanomagnetic microspheres are removed by magnetic separation to obtain separated and purified exosomes.

[0017] Wherein, the amino acid sequence of the temperature-sensitive specific polypeptide is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3.

[0018] It should be noted that the liquid containing exosomes refers to the liquid to be separated and purified from exosomes, which contains exosomes. For example, the liquid containing exosomes can be serum, serum-free cell culture medium supernatant, urine and other biological fluids. Washing can be done with physiological saline, and non-specific adsorbed substances refer to substances that are not related to exosome protein markers (CD63, CD9 and CD81, etc.).

[0019] In some optional embodiments, the incubation time at 2-8° C. is 1-12 hours.

[0020] In some optional embodiments, the incubation time at 25-37° C. is 10-15 minutes.

[0021] In some optional embodiments, modifying the nanomagnetic microspheres with a temperature-sensitive specific polypeptide comprises:

[0022] The polymer monomer, initiator and oleic acid-modified Fe3O4 nanoparticles are mixed to obtain epoxy polymer magnetic microspheres;

[0023] reacting the temperature-sensitive specific polypeptide with tri(2-carboxyethyl)phosphine (TCEP) to obtain a temperature-sensitive specific polypeptide in a thiol-reduced state;

[0024] The reduced thiol-reduced thermosensitive specific polypeptide is reacted with the epoxy polymer magnetic microspheres to obtain polypeptide-modified nanomagnetic microspheres.

[0025] In some optional embodiments, the polymer monomers include at least one of styrene and divinyl benzene, and also include at least one of glycidyl acrylate and glycidyl methacrylate. Preferably, the polymer monomers include styrene, divinyl benzene and glycidyl acrylate.

[0026] In some optional embodiments, the initiator is azobisisobutyronitrile.

[0027] In some optional embodiments, the temperature-sensitive specific polypeptide is obtained by the following steps:

[0028] Using exosome marker proteins CD63, CD9 or CD81 as target proteins, monoclonal phages displaying peptides were screened by phage display technology;

[0029] The monoclonal phages are incubated and bound to the exosome marker proteins CD63, CD9 or CD81 at 2-8°C, and then dissociated and eluted at 25-37°C. The eluted monoclonal phages are sequenced to screen positive monoclonal phages that bind to the exosome marker proteins CD63, CD9 or CD81 at 2-8°C and dissociate from the exosome marker proteins at 25-37°C. The terminal display polypeptide sequences of the positive monoclonal phages are obtained by sequencing nucleic acid substances;

[0030] The temperature-sensitive specific polypeptide is synthesized according to the terminal display polypeptide sequence of the positive monoclonal phage.

[0031] Due to the adoption of the above technical solution, the embodiments of the present invention have at least the following beneficial effects:

[0032] (1) Peptide-modified nanomagnetic microspheres can specifically bind to CD63, CD9 or CD81 proteins on the surface of exosomes, allowing the majority of exosome particles in body fluid samples to be separated, effectively overcoming the defects of conventional exosome enrichment techniques in the co-separation of impurities such as lipoproteins, and greatly improving the purity of exosomes;

[0033] (2) The method based on temperature difference specific non-destructive separation of exosomes is a specific non-destructive separation method. The separation conditions are mild and do not affect the integrity of the exosome morphology. It effectively overcomes the defect of conventional exosome enrichment technology that the polarity of the separation conditions causes damage to the exosomes.

[0034] (3) Peptide-modified nanomagnetic microspheres are combined with exosomes, and the affinity is regulated by low critical temperature. Exosomes can be quickly and easily separated by an external magnetic field, which is easy to operate. The peptide-modified nanomagnetic microspheres are removed by the external magnetic field, while the exosomes remain in the supernatant. The separation of exosomes and peptide-modified nanomagnetic microspheres can be successfully completed by solid-liquid separation, ensuring a high purity of exosomes.

[0035] (4) The price of peptides is low, which effectively overcomes the defects of conventional exosome enrichment technology, such as expensive instruments and high reagent costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The binding and dissociation effects of the temperature-sensitive specific polypeptides corresponding to SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3 with the exosome marker proteins CD63, CD9, and CD81 at 4°C and 37°C, respectively, are shown.

[0037] Figure 2 The binding effect of CD63 antibody to CD63 protein at 4°C and 37°C is shown.

[0038] Figure 3 Schematic diagram of the operational process for separating and purifying exosomes from biological fluid samples based on peptide-modified nanomagnetic microspheres.

[0039] Figure 4 The figure shows the difference in relative content of exosomal marker proteins CD63 and CD9 identified by WB under the same total protein amount of urine exosome samples separated by different exosome isolation methods.

[0040] Figure 5 Schematic diagram of the principle of detecting intact exosomes based on the immunosandwich method with antibodies to the same epitope as the exosome marker protein CD63.

[0041] Figure 6 The method based on temperature difference specific non-destructive separation of exosomes and the traditional separation method are shown. After separating equal amounts of biological fluid samples, the detection signal of intact exosomes is detected by ELISA immunosandwich using HRP-labeled anti-CD63 monoclonal antibody and immunomagnetic beads covalently coated with the antibody.

[0042] Figure 7 The total protein concentration in the separated fluid was measured by the BCA method after the same amount of biological fluid samples were separated by the method of temperature difference-based specific non-destructive separation of exosomes and the traditional separation method.

[0043] Figure 8 The ratio of the detection signal value of the intact exosome content in the separation fluid to the total protein concentration when isolating exosomes using the temperature difference-based specific non-destructive separation method and the traditional separation method is shown.

[0044] Fig. 9 The particle size of intact exosomes in the separation fluid is shown when separating exosomes based on temperature difference-specific non-destructive separation and traditional separation methods. DETAILED DESCRIPTION

[0045] The technical solution of the present invention will be described in detail below in conjunction with the specific implementation methods of the present invention. However, the following embodiments are only used to understand the present invention and cannot limit the present invention. The embodiments and features in the embodiments of the present invention can be combined with each other. The present invention can be implemented in a variety of different ways as defined and covered by the claims.

[0046] Table 2 Comparison table of amino acid name definitions

[0047]

[0048] The amino acids in the above amino acid sequences are natural L amino acids, or amino acid residue modifications, optical isomers (e.g., D-amino acids), or chemical molecule or group modifications (e.g., phosphorylation and dephosphorylation, acetylation and deacetylation, methylation and demethylation, adenylation and deadenylation, -SH and -SS- tautomerism, lipid group connection).

[0049] Example 1: Selection of phage-displayed polypeptides with temperature-regulated affinity

[0050] 1) Directed panning of phage-displayed peptide libraries

[0051] ① In the first round, polystyrene culture dishes were coated with exosome marker proteins CD63, CD9 and CD81 proteins overnight, with a concentration of 100 μg / mL, washed three times with 1 mL of phosphate buffered saline (PBST) containing 0.1% Tween 20, added with sodium bicarbonate solution dissolved with 1% bovine serum albumin (BSA), incubated at room temperature for 90 min, discarded the sample, washed three times with 1 mL of PBST, incubated with phage display peptide library for 1 hour, washed 10 times with PBST in an ice bath, then added with 150 μL PBS and incubated at 37°C for 1 h, 120 μL of liquid was taken out for titer determination and phage amplification;

[0052] ② In the second round, polystyrene culture dishes were coated with exosome marker proteins CD63, CD9 and CD81 proteins overnight, with a concentration of 10 μg / mL, washed three times with 1 mL of phosphate buffered saline (PBST) containing 0.1% Tween 20, added with sodium bicarbonate solution dissolved with 1% bovine serum albumin (BSA), incubated at room temperature for 90 min, discarded the sample, washed three times with 1 mL of PBST, incubated with phage display peptide library for 1 hour, washed 10 times with PBST in an ice bath, then added with 150 μL of PBS and incubated at 37°C for 1 h, 120 μL of liquid was taken out for titer determination and phage amplification;

[0053] ③ In the third round, polystyrene culture dishes were coated with exosome marker proteins CD63, CD9 and CD81 proteins overnight, with a concentration of 10 μg / mL, washed three times with 1 mL of phosphate buffered saline (PBST) containing 0.5% Tween 20, added with sodium bicarbonate solution dissolved with 1% bovine serum albumin (BSA), incubated at room temperature for 90 min, discarded the sample, washed three times with 1 mL of PBST, incubated with phage display peptide library for 1 hour, washed 10 times with PBST in an ice bath, then added with 150 μL PBS and incubated at 37°C for 1 h, took out 120 μL of liquid, performed titer determination, and picked monoclonal phage for identification.

[0054] 2) Identification of monoclonal phage specificity and temperature sensitivity

[0055] After the monoclonal phage was amplified, it was incubated and washed with sample wells coated with exosome marker proteins CD63, CD9 and CD81 at 4°C, and detected with horseradish peroxidase (HRP)-coupled Anti-M13 antibody; and a 37°C washing condition was set as a control group. Positive monoclonal phages with high specificity and obvious response to temperature changes (binding at 4°C and washing and dissociation at 37°C) were screened out; the positive monoclonal phage plasmids were sequenced by DNA, and the corresponding terminal display polypeptide sequences were obtained, namely VATDAHRRELKLGGGSC (SEQ ID No.1), TQVATAHLHSSHGGGSC (SEQID No.2) and SSAYRTGFFTHSGGGSC (SEQ ID No.3).

[0056] Example 2: Synthesis of temperature-sensitive specific polypeptides

[0057] 1) Weigh and swell the resin: Accurately weigh 0.1 g Wang-Resin (0.8 mmol / g), put it into a 20 mL sand core reactor, add three times the volume of the resin in dichloromethane (DCM), swell for 30 min, blow dry and set aside;

[0058] 2) Ninhydrin test: Take a small amount of resin, add ninhydrin solution, heat in an oil bath at 110℃, and the color of the solution remains unchanged within 5 minutes;

[0059] 3) Deprotection: Take three times the volume of the resin and react the deprotection solution (20% 4-methylpiperidine, dissolved in DMF) with the resin for 15 minutes, remove the deprotection solution, repeat once, and then wash with 2 times the volume of the resin. In the process of solid phase synthesis of active peptides, each time an amino acid is connected to complete a condensation reaction, the Fmoc protecting group of the amino acid at the end of the resin is removed, and then the next amino acid is condensed;

[0060] 4) Ninhydrin test: Take a small amount of resin, add ninhydrin solution, heat in an oil bath at 110℃, the solution should turn blue-purple within 5 minutes;

[0061] 5) Condensation: remove the cleaning solution, add the first protected amino acid (serine), then add the condensation reagent, dissolve it with a small amount of DMF, shake it thoroughly to mix and dissolve it, and react at room temperature for 30 minutes;

[0062] 6) Ninhydrin test: Take a small amount of resin and add ninhydrin solution. In an environment of 110℃, the color of the solution remains unchanged within 5 minutes.

[0063] 7) Amino acid addition: repeat the operations of step (3) and step (6), and add the protected amino acids one by one according to the target polypeptide sequence (SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3);

[0064] 8) Chain termination and elution: When all amino acids are connected into a complete peptide chain in the predetermined order, wash the resin with methanol 3 to 4 times and then add the cutting solution. Oscillate at room temperature for 2.5 hours, take the filtrate, add ice petroleum ether (stored at -20℃), stir magnetically, centrifuge at 5000rpm, remove the supernatant, resuspend, repeat the operation three times, and finally dry it with a vacuum reactor to obtain the target temperature-sensitive specific polypeptide.

[0065] Example 3: Identification of specificity and temperature difference affinity of temperature-sensitive specific polypeptides

[0066] Determine the specific affinity of the temperature-sensitive specific peptide to the target protein under different conditions to verify whether the peptide meets the temperature-sensitive specific binding and dissociation capabilities of the target protein. The steps are as follows:

[0067] 1) Dissolve the exosome marker proteins CD63, CD9 and CD81 in 0.1 M sodium bicarbonate solution at 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.13 μg / mL, 1.56 μg / mL and 0 μg / mL, respectively, coat the wells of polystyrene plates at 4°C overnight, then block with 1% BSA dissolved in 0.1 M sodium bicarbonate solution at 37°C for 2 hours, and wash three times with PBST;

[0068] 2) 5 μg / mL of the temperature-sensitive specific peptides corresponding to SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3 and the horseradish peroxidase-coupled complex were added to the exosome marker protein-coated wells at 100 μL and reacted at 4°C for 1 hour. Two groups were set up, and a control group of CD63 antibody horseradish peroxidase-coupled complex and CD63 protein reaction was set up at the same time. After the reaction, the cells were washed three times with PSBT at 4°C and 37°C respectively;

[0069] 3) Add 150 μL TMB substrate solution and incubate in the dark for 10 minutes, then add 50 μL 2M HCl solution to terminate the reaction and detect the absorbance at 450 nm.

[0070] like Figure 1As shown, the temperature-sensitive specific peptides corresponding to SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3 have significant specificity with the exosome marker proteins CD63, CD9, and CD81, and also exhibit significant affinity temperature sensitivity. After the temperature-sensitive specific peptides bind to the target protein at 4°C, the detection signal still remains high when using a 4°C cleaning solution, reaching more than 2.0. However, after washing with a 37°C PBST cleaning solution, the signal value drops sharply, indicating that the temperature rises and the peptide dissociates from the target protein. Figure 2 As shown, the CD63 antibody control group showed that the binding effect after washing at 37°C was better than that under the washing condition of 4°C, which is consistent with the situation that traditional antigen-antibody requires a certain temperature to have a better binding effect.

[0071] Example 4: Preparation of polypeptide-modified nanomagnetic microspheres

[0072] 1) Preparation of epoxy polymer magnetic microspheres

[0073] 1.5 g styrene, 0.5 g divinylbenzene, 0.5 g glycidyl acrylate, 3.0 g oleic acid-modified Fe3O4 superparamagnetic nanoparticles, and 2 g polyvinyl pyrrolidone were dissolved in 100 mL of 90% ethanol aqueous solution, mechanically stirred at 800 rpm for 2 hours, then nitrogen was passed for 30 minutes, and then 1 g azobisisobutyronitrile as an initiator was added, and the mixture was reacted in an oil bath at 80°C for 24 hours. The mixture was adsorbed in a magnetic field and washed alternately with ethanol and water three times to obtain epoxy polymer magnetic microspheres.

[0074] 2) Preparation of peptide-modified nanomagnetic microspheres

[0075] ① Incubate 10 mg of the temperature-sensitive specific peptide to be modified with 5 mM TCEP at room temperature for 10 minutes to reduce the disulfide bonds between the peptide molecules to sulfhydryl groups, and then desalt the solution through a desalting column and replace it with a 0.05 M Tris-HCl buffer solution at pH 8.4 for later use;

[0076] ② Wash 1 g of epoxy polymer magnetic microspheres three times with 0.05 M Tris-HCl buffer solution at pH 8.4, add 10 mg of desalted thermosensitive peptide solution, invert and mix at 37°C for 24 hours, then block with 0.05 M Tris-HCl buffer solution containing 50 mM cysteine, invert and mix at 37°C for 24 hours, then wash with PBST to obtain polypeptide-modified nanomagnetic microspheres, which are stored in PBS solution for later use.

[0077] Example 5: Separation and purification of urine exosomes using peptide-modified nanomagnetic microspheres

[0078] By using low critical temperature to regulate the affinity of temperature-sensitive specific peptides, the peptide-modified nanomagnetic microspheres specifically bind to exosomes in urine at 4°C, separate and capture exosomes from the liquid phase, and release exosomes from the microspheres at 37°C, thereby obtaining high-purity, non-damaged exosomes, such as Figure 3 As shown, the specific steps are as follows:

[0079] 1) Urine sampling and pretreatment: Take a midstream urine sample of the morning urine, centrifuge it at 4°C and 500 x g for 10 min, take the supernatant and discard the precipitate; centrifuge it again at 4°C and 2000 x g for 10 min, take the supernatant and discard the precipitate; centrifuge it again at 4°C and 10000 x g for 30 min, take the supernatant and discard the precipitate, and filter it through a 0.22 μm filter to obtain a pretreated urine sample;

[0080] 2) Isolation of exosomes: Take 10 mL of pretreated urine sample, add 20 mg of peptide-modified nanomagnetic microspheres to the pretreated urine sample, invert and mix at 4°C for 1 hour, perform magnetic adsorption on ice, remove the supernatant, wash three times with 10 mL of 4°C precooled PBS, and finally add 200 μL of PBS solution, vortex to disperse the peptide-modified nanomagnetic microspheres, treat in a 37°C water bath for 10 min, use a magnetic stand for adsorption, take the supernatant, and obtain a temperature-difference-specific non-destructively separated and purified exosome solution.

[0081] In order to compare the effects of the separation method in this embodiment and the separation method in the prior art, the following control group was set up:

[0082] ① Ultracentrifugation method: Take 10 mL of pretreated urine sample and centrifuge it at 4°C and 100,000 x g for 1 hour, remove the supernatant, and resuspend the precipitate with 10 mL PBS; centrifuge it again at 4°C and 100,000 x g for 1 hour, and resuspend the precipitate with 200 μL PBS solution to obtain the exosome solution;

[0083] ② PEG precipitation method: Use 0.1M NaCl in PBS to prepare 50% PEG 8000 solution, take 2.5 mL and put it into 10 mL pretreated urine sample, vortex mix, and stand at 4°C overnight; then centrifuge at 4°C and 10,000 xg for 30 minutes, remove the supernatant, resuspend the precipitate with 1 mL 10% PEG 8000 PBS solution, and stand on ice for 1 hour; then centrifuge at 4°C and 10,000 xg for 30 minutes, resuspend the precipitate with 200 μL PBS solution to obtain exosome solution;

[0084] ③ Immunomagnetic affinity separation method: 100 μL Dynabeads® CD63 +Exosome separation magnetic beads were added to 10 mL of pretreated urine sample and incubated overnight at 4°C with inversion. The supernatant was removed by magnetic adsorption, and the magnetic beads were washed three times with PBST and once with PBS. Then, 100 μL of 0.1 M Gly-HCl buffer with a pH of 2.2 was added to resuspend the magnetic beads and incubated at room temperature for 10 min. Then, 0.1 M Tris-HCl buffer solution with a pH of 8.6 was used for neutralization to obtain purified exosomes.

[0085] The urine exosome samples separated by the above different methods were subjected to Western blotting (WB) experiments with a total protein of 4 μg to identify the CD63 protein concentration. The results are as follows Figure 4 As shown, the method of the present invention has the highest specificity compared with the immunomagnetic bead affinity separation method. At the same time, the exosomes contain CD9, indicating that it is the exosomes that are bound, not the simple CD63 protein.

[0086] Example 6: Separation and purification of exosomes from cell culture supernatant using peptide-modified nanomagnetic microspheres

[0087] The affinity of thermosensitive specific peptides is regulated by low critical temperature. The peptide-modified nanomagnetic microspheres are specifically bound to exosomes in the cell culture supernatant at 4°C, and the exosomes are separated and captured from the liquid phase. The microspheres release the exosomes at 37°C, thereby obtaining high-purity, non-damaged exosomes. The specific steps are as follows:

[0088] 1) Pretreatment of cell culture supernatant samples: Take the cell culture supernatant sample of serum-free culture. Centrifuge at 4°C, 300 xg for 10 min, take the supernatant, and remove free cells; centrifuge again at 4°C, 2000 xg for 10 min, take the supernatant, and remove large cell debris; centrifuge again at 4°C, 10000 xg for 30 min, take the supernatant, remove large vesicles and cell debris, and filter through a 0.22 μm filter to obtain the pretreated cell culture supernatant sample;

[0089] 2) Isolation of exosomes: Take 10 mL of pretreated cell culture supernatant sample, add 20 mg of peptide-modified nanomagnetic microspheres to the pretreated cell culture supernatant sample, invert and mix at 4°C for 1 hour, perform magnetic adsorption on ice, remove the supernatant, wash three times with 10 mL of 4°C precooled PBS, finally add 200 μL of PBS solution, vortex to disperse the peptide-modified nanomagnetic microspheres, treat in a 37°C water bath for 10 min, use a magnetic stand for adsorption, and take the supernatant to obtain the temperature difference specific non-destructive separation and purification of exosome solution.

[0090] In order to compare the effects of the separation method in this embodiment and the separation method in the prior art, the following control group was set up:

[0091] ① Ultracentrifugation method: Take 10 mL of the pre-treated cell culture supernatant sample, centrifuge it at 4°C and 100,000 x g for 1 hour, remove the supernatant, and resuspend the precipitate with 10 mL PBS; centrifuge it again at 4°C and 100,000 x g for 1 hour, and resuspend the precipitate with 200 μL PBS solution to obtain the exosome solution;

[0092] ② PEG precipitation method: Prepare 50% PEG 8000 solution with 0.1M NaCl in PBS, take 2.5 mL and put it into 10 mL pre-treated cell culture supernatant sample, vortex to mix, and stand at 4°C overnight; then centrifuge at 4°C and 10,000 xg for 30 minutes, remove the supernatant, resuspend the precipitate with 1 mL 10% PEG 8000 PBS solution, and stand on ice for 1 hour; then centrifuge at 4°C and 10,000 xg for 30 minutes, resuspend the precipitate with 200 μL PBS solution to obtain exosome solution;

[0093] ③ Immunomagnetic affinity separation method: 100 μL Dynabeads® CD63 + Exosome separation magnetic beads were added to 10 mL of pretreated cell culture supernatant sample, incubated overnight at 4°C with inversion, and the supernatant was removed by magnetic adsorption. The magnetic beads were washed three times with PBST and once with PBS. Then, 100 μL of 0.1 M Gly-HCl buffer with a pH of 2.2 was added to resuspend the magnetic beads and incubated at room temperature for 10 min. Then, 0.1 M Tris-HCl buffer solution with a pH of 8.6 was used for neutralization to obtain purified exosomes.

[0094] Example 7: Separation and purification of exosomes from peripheral blood using peptide-modified nanomagnetic microspheres

[0095] The affinity of thermosensitive specific peptides is regulated by low critical temperature. At 4°C, the peptide-modified nanomagnetic microspheres specifically bind to exosomes in peripheral blood, separate and capture exosomes from the liquid phase, and release exosomes from the microspheres at 37°C, thereby obtaining high-purity, non-damaged exosomes. The specific steps are as follows:

[0096] 1) Peripheral blood sample extraction and pretreatment: Use a sampling tube containing an anticoagulant (such as sodium citrate or EDTA-2Na) to collect peripheral blood, centrifuge at 20°C and 2500 xg for 15 min, take the supernatant above the blood cell layer, and remove red blood cells and platelets; centrifuge again at 20°C and 2500 xg for 15 min, take the supernatant, remove large cell fragments, and obtain a sample without platelets and red blood cells;

[0097] 2) Isolation of exosomes: Take 10 mL of pretreated plasma sample, add 20 mg of peptide-modified nanomagnetic microspheres to the pretreated plasma sample, invert and mix at 4°C for 1 hour, perform magnetic adsorption on ice, remove the supernatant, wash three times with 10 mL of 4°C precooled PBS, and finally add 200 μL of PBS solution, vortex to disperse the peptide-modified nanomagnetic microspheres, treat in a 37°C water bath for 10 min, use a magnetic stand for adsorption, take the supernatant, and obtain a temperature-difference-specific non-destructively separated and purified exosome solution.

[0098] In order to compare the effects of the separation method in this embodiment and the separation method in the prior art, the following control group was set up:

[0099] ① Ultracentrifugation method: Take 10 mL of pretreated plasma sample, centrifuge at 4°C and 100,000 x g for 1 hour, remove the supernatant, and resuspend the precipitate with 10 mL PBS; centrifuge again at 4°C and 100,000 x g for 1 hour, resuspend the precipitate with 200 μL PBS solution to obtain purified peripheral blood exosomes;

[0100] ② PEG precipitation method: Prepare 50% PEG 8000 solution with 0.1M NaCl in PBS, take 2.5 mL and put it into 10 mL pretreated plasma sample, vortex mix, and stand at 4°C overnight; then centrifuge at 4°C and 10,000 xg for 30 minutes, remove the supernatant, resuspend the precipitate with 1 mL 10% PEG 8000 PBS solution, and stand on ice for 1 hour; then centrifuge at 4°C and 10,000 xg for 30 minutes, resuspend the precipitate with 200 μL PBS solution to obtain purified peripheral blood exosomes;

[0101] ③ Immunomagnetic affinity separation method: 100 μL Dynabeads® CD63 + Exosome separation magnetic beads were added to 10 mL of pretreated plasma sample and incubated overnight at 4°C with inversion. The supernatant was removed by magnetic adsorption, and the magnetic beads were washed three times with PBST and once with PBS. Then, 100 μL of 0.1 M Gly-HCl buffer with a pH of 2.2 was added to resuspend the magnetic beads and incubated at room temperature for 10 min. Then, 0.1 M Tris-HCl buffer solution with a pH of 8.6 was used for neutralization to obtain purified peripheral blood exosomes.

[0102] Example 8: Identification of the content and purity of structurally intact exosomes

[0103] The same CD63 monoclonal antibody is used as both a solid-phase binding antibody and a detection antibody to form a sandwich immune complex with exosomes. Exosome cells will only appear when the exosome structure is complete and there are more than two exosome marker proteins CD63. The principle diagram is shown in the figure below. Figure 5 As shown, the content of intact exosomes was evaluated by signal strength. The purity was evaluated by comparing the total protein concentration of exosomes extracted by different methods with the ratio of exosome CD63 detection signals. The specific steps for obtaining the CD63 detection signal ratio are as follows:

[0104] 1) Dissolve CD63 antibody at 10 μg / mL in 0.1 M NaHCO3 solution at pH 8.6, coat the plate overnight in a 96-well plate, wash three times with PBST, and then add 1% BAS dissolved in 0.1 M NaHCO3 solution at pH 8.6 for complete blocking;

[0105] 2) Take urine, cell culture supernatant, and peripheral blood exosomes separated by different separation methods, dilute them in half with PBS, and then filter them with a 0.22 μm filter. Then add 50 μL of the exosome sample to be tested to each well. After incubation at 37°C for 30 minutes, add 50 μL of CD63-HRP complex and continue to react for 1 hour; then wash three times with PBST, add 150 μL of TMB substrate solution for color development, incubate at 25°C in the dark for 10 minutes, add 50 μL of 2 M H2SO4 solution for termination, and use an enzyme reader to read the signal value at 450 nm to obtain the content information of complete exosomes separated by different separation methods.

[0106] like Figure 6 As shown in the figure, the exosome content obtained by PEG precipitation method is relatively high; the content of the method based on temperature difference specific separation of exosomes (the method of the present invention) is second; the content of ultracentrifugation method is relatively low; the complete exosome signal obtained by immunomagnetic bead affinity separation method is also relatively weak, which is consistent with the attached Figure 4 The correlation between the WB results was poor, indicating that the structure of the exosomes was destroyed after acid washing.

[0107] The steps to obtain exosome purity information are as follows:

[0108] 1) Add 180 μL of BCA working solution reagent to a 96-well plate, then add 20 μL of the exosome sample to be tested (urine, cell culture supernatant, peripheral blood) and the standard concentration working product, shake and mix, and incubate at 37°C for 30 minutes to allow the reaction to proceed fully;

[0109] 2) Use an ELISA reader to read the absorbance at 562 nm, establish a standard curve based on the concentration of the standard and the absorbance, and then calculate the total exosome protein concentration information based on the standard curve;

[0110] 3) Divide the exosome content signal value by the total protein concentration value to obtain the exosome purity information.

[0111] like Figure 7As shown, the total protein concentration of exosomes separated from various samples by PEG precipitation method is relatively high; followed by the total protein concentration obtained by ultracentrifugation method; the total protein concentration obtained by the method based on temperature difference specific non-destructive separation of exosomes (the method of the present invention) is close to that obtained by the immunomagnetic bead affinity separation method.

[0112] like Figure 8 As shown, the results show that the method based on temperature difference specific non-destructive separation of exosomes (the method of the present invention) has the highest purity of intact exosomes, indicating that this separation method has unique advantages over ultracentrifugation, PEG precipitation and immunomagnetic bead affinity separation, taking into account the advantages of exosome integrity, purity, and content.

[0113] like Fig. 9 As shown, the method based on temperature difference specific non-destructive separation of exosomes (the method of the present invention) obtained a standard exosome size of about 115 nm, which is close to the exosome particle size of the ultracentrifugation method; however, the exosome particle size of the PEG precipitation method was significantly increased, which may be due to aggregation caused by polymer-induced precipitation.

[0114] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same or equivalent means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. A temperature-sensitive specific polypeptide, characterized in that: The amino acid sequence of the temperature-sensitive specific polypeptide is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.

3.

2. Use of the temperature-sensitive specific polypeptide according to claim 1 in isolating exosomes.

3. A method for non-destructive separation of exosomes based on temperature difference specificity, characterized in that: The following steps are involved: Modifying nanomagnetic microspheres with temperature-sensitive specific polypeptides to obtain polypeptide-modified nanomagnetic microspheres; The liquid containing exosomes is mixed with the polypeptide-modified nanomagnetic microspheres, and incubated at 2-8°C to allow the exosomes to bind to the polypeptide-modified nanomagnetic microspheres. Under the action of a magnetic field, the polypeptide-modified nanomagnetic microspheres capture and separate the exosomes from the liquid, and non-specific adsorbed substances are removed by washing. Thereafter, the liquid is incubated at 25-37°C to allow the exosomes to dissociate from the polypeptide-modified nanomagnetic microspheres, and the polypeptide-modified nanomagnetic microspheres are removed by magnetic separation to obtain separated and purified exosomes. Wherein, the amino acid sequence of the temperature-sensitive specific polypeptide is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.

3.

4. The method according to claim 3, characterized in that The incubation time at 2-8°C is 1-12 hours.

5. The method according to claim 3, characterized in that: The incubation time at 25-37°C is 10-15 minutes.

6. The method according to claim 3, characterized in that Modification of nanomagnetic microspheres with temperature-sensitive specific peptides includes: The polymer monomer, initiator and oleic acid-modified Fe3O4 nanoparticles are mixed to obtain epoxy polymer magnetic microspheres; reacting the temperature-sensitive specific polypeptide with tris(2-carboxyethyl)phosphine to obtain a temperature-sensitive specific polypeptide in a thiol-reduced state; The thermosensitive specific polypeptide in the thiol-reduced state is reacted with the epoxy polymer magnetic microspheres to obtain polypeptide-modified nano magnetic microspheres.

7. The method according to claim 6, characterized in that The polymer monomers include at least one of styrene and divinylbenzene, and also include at least one of glycidyl acrylate and glycidyl methacrylate.

8. The method according to claim 7, characterized in that The polymer monomers include styrene, divinyl benzene and glycidyl acrylate.

9. The method according to claim 6, characterized in that The initiator is azobisisobutyronitrile.

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